This invention relates to methods for purifying and activating proteins that are produced as insoluble, biologically inactive inclusion bodies in microorganisms that have been transformed with recombinant DNA expression vectors to direct expression of the protein of interest.
Recombinant DNA technology allows the insertion of a vector carrying foreign (heterologous) DNA into a microorganism in a manner which allows the heterologous DNA to be expressed; that is, the vector contains genetic instructions which direct the microorganisms to produce a protein which is encoded by a portion of the heterologous DNA sequence. By growing transformant microorganisms in a fermentor and subjecting them to conditions under which the heterologous DNA is expressed, valuable proteins can be produced in large quantity at relatively low cost.
Unfortunately, many heterologous proteins which are produced in transformant microorganisms do not fold into their native three-dimensional conformation in the host cell environment. This improper folding of the expressed protein has several untoward consequences. In the first place, the improperly folded proteins tend to form aggregates which are insoluble within the host cell. These insoluble aggregates are recognizable within the cell as "inclusion bodies", sometimes also referred to as "refractile bodies" and/or "protein granules." The formation of inclusion bodies may also be partially caused by oligomerization of the protein, that is, the formation of covalent intermolecular disulfide bonds. Not only are the improperly folded proteins insoluble, but also they are biologically inactive. As exemplary of heterologous proteins which form insoluble, biologically inactive inclusion bodies upon expression in a host cell, one can mention animal growth hormones and growth factors such as bovine growth hormone, swine growth hormone and somatomedin.
In order to produce useful proteins, it is necessary to convert the improperly folded inclusion body proteins into their native conformations, in which they are soluble and biologically active. Moreover, it is necessary to purify the protein in order to remove contaminating cell debris and host cell proteins. A number of schemes have been proposed for converting inclusion body proteins into their soluble, native configurations. Unfortunately, these schemes are often incompatible with protein purification procedures, such as ion-exchange chromatography. The conditions of purification tend to inhibit the ability to maintain the protein in solution, often resulting in a substantial loss of protein due to reaggregation and precipitation. Consequently, most of the schemes proposed for recovering proteins from inclusion bodies in purified, soluble, biologically active form have resulted in very low yields of the protein produced by the microorganisms.
U.S. Pat. No. 4,511,503 discloses a typical scheme for recovering proteins from inclusion bodies in transformant microorganisms. The inclusion body proteins are treated with a strong denaturant, which causes the improperly folded protein molecules to unfold and become soluble. The denaturant is subsequently removed, for example, by dialysis, in order to allow the protein to refold into its native conformation. The most commonly employed strong denaturant in schemes of this type has been guanidine hydrochloride.
U.S. Pat. No. 4,511,502 discloses a similar process wherein the solubilized protein/denaturant solution is passed over a molecular sieve or centrifuged at high speed to remove higher molecular weight components.
U.S. Pat. No. 4,518,526 also discloses a similar process. In this process, the transformant cell culture is treated with a buffered solution of sufficient ionic strength to solubilize most of the host cell protein, whereas the heterologous protein remains insoluble. The cells are then lysed, the supernatant containing the solubilized host cell protein removed and the insoluble inclusion bodies solubilized in the strong denaturant.
Other publications disclosing denaturation/renaturation schemes for converting inclusion body proteins into their soluble, native conformations include PCT publication WO No. 83/04418, European Patent Application Publication No. 0 123 928, European Patent Application Publication No. 0 121 775, European Patent Application Publication No. 0 116 778 and European Patent Application Publication No. 0 114 507.
As previously indicated, most of the proposed denaturation/renaturation schemes have employed guanidine hydrochloride as the denaturant. While guanidine hydrochloride is characterized by an excellent ability to solubilize inclusion body proteins, its use entails some problems. Upon dialysis against denaturant-free buffer to remove the guanidine hydrochloride, a substantial amount of the solubilized protein reaggregates, apparently due to improper refolding. Moreover, when guanidine-solubilized protein is purified by methods such as ion-exchange chromatography--which are necessary to obtain the degree of purity required in the final product--very substantial losses of protein are incurred due to reaggregation. Fouling and plugging of the column tends to occur in an inordinately short time, severely limiting the useful life of the column. Typically, we have found that guanidine solubilization of bovine growth hormone inclusion bodies, followed by ion-exchange chromatography yielded only 4-12% product recovery.
Another major problem associated with the use of guanidine hydrochloride--one which is particularly important from a commercial production standpoint--is its high cost. It would be highly desirable to employ a solubilizing agent which is comparable to guanidine hydrochloride in its ability to solubilize inclusion body proteins, but without the associated high cost of guanidine. U.S. Pat. No. 4,511,503 suggests the use of detergents such as sodium dodecyl sulfate (SDS) as denaturants. However, there is no demonstration of its use, nor is there a proposal for a method to remove the detergent from the protein and purify the protein.
Detergents such as SDS are highly effective denaturing agents. Moreover, SDS is a much less expensive reagent than guanidine hydrochloride. Accordingly, its potential use in recovering inclusion body proteins is attractive from the standpoint of commercial production economics. Compared with guanidine hydrochloride, however, SDS binds to the denatured protein much more tightly, making its complete removal from the protein problematical.
O. H. Kapp and S. W. Vinogradov demonstrated that SDS could be removed from several proteins by chromatography on the ion-retardation resin AG11A8 (Anal. Biochem., 91:230-233 [1978]). It was said that from 0.1 to 1.4 moles of SDS remained on each mole of protein treated in this manner. K. Weber and D. J. Kuter demonstrated that SDS could be removed from aspartate transcarbamylase by incubation in urea and subsequent anion-exchange chromatography (J. Biol. Chem., 246:4504-4509 [1971]). In both instances, however, the starting proteins were in pure, biologically active form. There is no suggestion of a method for efficiently recovering protein in a pure, biologically active form from insoluble, intracellular inclusion bodies.